Compounds and use thereof to modify transport across cell membranes

ABSTRACT

The invention provides the use of a compound capable of preferential interaction with plasma membrane lipid microdomains (PMLMs) as enhancers of transport processes across endothelial, epithelial and mesothelial membranes (ie including blood-brain-blood barrier and gastrointestinal mucosal membranes). When associated with therapeutic agents, the compounds act as transport vehicles. When the compounds interact with PMLMs in such a way as to inhibit transport across the membrane, the compounds function as anti-infective agents.

This invention relates generally to the field of drug delivery, and inparticular to methods and compositions for aiding the delivery andaction of physiologically active agents, and to novel compounds usefulin such methods and compositions.

The invention relates particularly to molecules that interact in aspecific manner with endothelial and epithelial cell membrane surfacesso as to control the interactions (including cell membrane penetration)of macromolecules and cellular components through specific cell membranemediated processes related to the formation and activity oflipid/protein microdomains. Such molecules are termed biovectors andenhancers.

In recent years, a number of structural elaborations have been added tothe classical Singer & Nicholson fluid mosaic model of membranestructure in order to accommodate new experimental data directed towardsthe properties of the lipids and so-called ‘detergent-insoluble’membrane components. These views are embodied in a recent review articleby Simons & Ikonen (Nature (1997) 387, 569–572) with the clear statementthat some lipidic membrane components may phase-separate and togetherwith membrane proteins form micro-domains that exist as ‘raft’-likestructures ‘afloat’ within the more familiar, fluid phospholipidbilayer. Such structures are referred to herein as “plasma membranelipid microdomains” (PMLMs). This process seems to be promoted by thepresence of cholesterol and some sphingolipids. PMLMs equipped withcholesterol and other exotic lipids may present a very differentenvironment to that of the more numerically common glycerophospholipids.

Cellular membranes contain lipids which represent an environment formembrane associated proteins. The lipids are asymmetrically distributedthrough the membrane giving a degree of order to the membrane structure.Further, the lateral packing of sphingolipids and cholesterol producesmoving platforms or PMLMs into or onto which specific proteins becomeattached. These proteins can be included or excluded by these lipidmicrodomains in a specific manner. It is believed that thesemicrodomains act as transport centres for macromolecular trafficking andfor intracellular signalling.

Glycosphingolipids and cholesterol are also associated with caveolae.These are non coated invaginations/vesicles involved in endocytotic andtranscytotic processes and, therefore, give credence to the raftmicrodomains also being involved in membrane trafficking, albeit at anearlier stage. These microdomains act as centres for the sorting anddistribution of proteins in the membrane and form a focal point forsignalling events and surface interactions.

The site of action of therapeutically active agents and the site towhich they are administered generally do not coincide. In order for theactive agent to reach its intended site of action it generally mustcross one or more cellular (endothelial or epithelial) barriers. Inorder to achieve therapeutically effective levels of the active agent atits active site, particularly in the face of competing naturaldegradation and clearance processes, it may therefore be necessary toadminister excessively large quantities of the drug. For these reasons,there is clearly a need for improved mechanisms for facilitating thedelivery of therapeutic agents across cell membranes.

Agents currently in use for this purpose include a number of peptides orchemical compounds that have been produced to interact specifically withreceptors on membranes. The present invention, however, relates to thetargeting of biovectors that interact with components (includingspecific receptors and lipids) of lipid microdomains in a specificmanner to subvert existing transport processes to either enhance drugdelivery or inhibit infection at the cellular level.

This invention is based on the discovery that certain compoundsincluding peptides and peptide mimetics interact preferentially withPMLMs. As PMLMs are believed to be involved with biosynthetic andendocytotic trafficking and signal transduction, these compounds maythus enhance these processes and inhibit or control transport andinfective processes.

The invention thus provides the use of a compound capable ofpreferential interaction with PMLMs as enhancers of transport processesacross endothelial, epithelial and mesothelial membranes (ie includingblood-brain-blood barrier and gastrointestinal mucosal membranes). Whenassociated with therapeutic agents, the compounds act as transportvehicles. When the compounds interact with PMLMs in such a way as toinhibit transport across the membrane, the compounds function asanti-infective agents.

The invention also provides a pharmaceutical composition comprising atleast one therapeutically active agent and a compound capable ofpreferential interaction with PMLMs.

According to another aspect of the invention there is provided aconjugate of a therapeutically active agent with a compound capable ofpreferential interaction with PMLMs.

The invention further provides a method of enhancing transport across anendothelial, epithelial or mesothelial cellular membrane, which methodcomprises administering to the membrane a compound capable ofpreferential interaction with PMLMs. An analogous aspect of theinvention provides a method of inhibiting infection by administering tothe membrane a compound capable of preferential interaction with PMLMsso as to inhibit transport across the membrane.

Membranes across which transport may be either enhanced or inhibited bythe methodology of the invention include pulmonary epithelial membranes,gastrointestinal mucous membranes and the blood-brain barrier.

Many compounds useful in the practice of the invention are believed tobe novel and these compounds per se represent a further feature of theinvention. The compound referred to below as BVS1 has been disclosed inWO 99/36092 as the means to block certain membrane interactionsassociated with specific viral infections. The present inventionrepresents a generic technique for drug delivery and otheranti-infective processes associated with PMLM structures.

Preferred agents to be used in this invention are a series of peptidesand peptide mimetics that interact with lipid microdomain components andthose comprising at least one negatively charged entity together with ahydrophobic moiety, and also similar peptides or mimetics to whichlipids such as ketocholestanol, cholesterol or a similar sterol isattached.

One group of compounds which are of utility in, and represent a furtheraspect of, the present invention is represented by the general formulaBVS-Gen1:BVS-Gen1:POS-Pol₁-HYD-Pol₂-NEGin which

-   -   POS represents an amino acid residue including the N-terminal        positive charge,    -   Pol₁ represents a short sequence of polar, possibly including        positively charged, residues,    -   HYD represents a hydrophobic amino acid sequence (ie more than        one and typically up to five residues), optionally substituted        or interrupted by one or more polar groups,    -   Pol₂ represents a short sequence of polar, possibly including        positively charged, residues, and    -   NEG represents a negatively charged amino acid residue including        the carboxylate terminus.

Active sequences typically do not include proline and occur with apreference for the charged/polar moieties located towards the termini.

The sequence Pol₁ typically contains up to 8 residues, and typicallyfrom 3 to 5 residues. Amino acids that may be included in the sequencePol₁ are illustrated in the examples given below.

Amino acids that may be included in the sequence HYD are illustrated inthe specific examples given below.

Amino acids that may be included in the sequence Pol₂ typically includethe sequences illustrated in the list below.

Particular amino acid residues which NEG may represent are aspartic acidand glutamic acid.

Specific examples of molecular structures which exhibit the desiredproperties are those with the formulae denoted BVS1–BVS17:

PEPTIDE SEQUENCE BVS1 AVGIGALFLGFLGAAG BVS2 GVFVLGFLGFLA BVS3GFLGFLAVVFLG BVS4 FVLGFVGLFLGA BVS5 GVFVLGLFGFLA BVS6 GVFVLFGLGFLA BVS7GVAVLGALGFLA BVS8 GVAVLGFLGALA BVS9 GVFVLGFLGFLATAGS BVS10GVFVLFLGFLATAGSAMGAASLTV BVS11 GVFVLGFLGFLATAGSAMGAASLT BVS12GVFVLGFLGFLTTAGAAMGAASLT BVS13 GVLVLGFLGFLTTAGAAMGAASLT BVS14AIGALFLGFLGAAG BVS15 AGALFLGFLGAAG BVS16 AGIGALFLGFLGAAG BVS17GVFVGFLGFLTTAGAAMGAASLTL

With the exception of BVS1, such compounds are believed to be novel andrepresent a further aspect of the invention.

Another group of compounds which may be useful are those comprising anyof BVS1–BVS16 with one or more negatively or positively charged terminalgroups, ie compounds of the general formula BVS-Gen2:BVS-Gen2:A-SEQ-Bwherein

-   -   SEQ represents any one of the sequences BVS1–BVS16, and    -   at least one of A and B represents one or more negatively or        positively charged terminal amino acid residues.

Examples of amino acid residues that A and/or B may represent areaspartic acid, glutamic acid and lysine.

Examples of compounds of general formula BVS-Gen2 are those denotedBVS18–BVS21:

BVS18 DAVGIGALFLGFLGAAGD BVS19 DDAVGIGALFLGFLGAAGDD BVS20EAVGIGALFLGFLGAAGE BVS21 EAVGIGALFLGFLGAAGK

A still further group of compounds which may be useful are conjugates ofany of the above compounds BVS-Gen1, BVS1–BVS17 and BVS-Gen2 with amembrane-compatible lipid. Examples of such lipids include sterols, egketocholestanol and cholesterol, and other compounds with similarproperties (eg sphingolipids and so-calledglycosylphosphatidylinositol(gpi)-anchors) that may be used to targetPMLMs. These covalent adjuncts are preferably located towards thetermini but may also function if located within the body of thesequence.

In all of the above formulae, the letters A, D, E, F, G etc have theconventional meanings used to represent amino acids, as follows:

-   A alanine-   D aspartic acid-   E glutamic acid-   F phenylalanine-   G glycine-   I isoleucine-   K lysine-   L leucine-   M methionine-   S serine-   T tyrosine-   V valine

Formulations for administration of such materials are well known and canbe used in this invention. Routes of administration include, by way ofexample only, parenteral, inhalation (dry powder, aerosol), buccal andtransdermal. Microcapsules, liposomes etc may be used to deliver thematerials to endothelial or epithelial surfaces in a specific manner.Enterically coated capsules and the like may be used forgastrointestinal delivery. The materials may also be administeredtopically, eg using needleless injectors etc. The amount to beadministered depends on factors routinely considered by those skilled inthe art and can be determined by routine experimentation.

The invention will now be described in greater detail, by way ofillustration only, with reference to the following Examples and theaccompanying Figures, in which

FIG. 1 shows the interaction of designer peptides with human cellspossessing membrane receptors;

FIG. 2 shows the interaction of designer peptides with purified isolatedreceptor;

FIG. 3 shows the interaction of peptido-mimetic with purified isolatedreceptor; and

FIG. 4 shows schematically apparatus used to measure transport across acellular membrane.

The following Examples illustrate the behaviour of various peptides andother molecules that promote transcellular transport of therapeuticmolecules etc. These studies make use of techniques that show theinteractions of molecules with receptors and cell membranes. Briefly,the time evolution and overall extent of the interactions of many typesof molecule with membranes, including proteins and peptides, have beenshown to be accessible using a technique recently introduced in ourlaboratory. Measurements rely on the fact that most proteins thatinteract with membranes possess a net charge and once bound, result inchanges of the electrostatic potential present on the membrane surface.We have shown that changes of the surface electrostatic potentialinfluence the fluorescence yield of a class of fluorescent phospholipidsof which fluoresceinphosphatidylethanolamine (FPE) has been shown to beparticularly versatile and reliable in a number of systems. FPE may beadded to membranes in amounts that do not affect any of the propertiesof the membrane (1 mol FPE:800–1,000 mol phospholipidlipid) with thefluorescent indicator moeity precisely located at the membrane-solutioninterface. The underlying theory and application of this technique isoutlined in:

-   Wall, J., Ayoub, F. & O'Shea. P. (1995) J. Cell Sci. 108, 2673–2682-   Cladera, J. and O'Shea, P. (1998) Biophys. J. 74, 2434–2442-   Cladera, J., Martin, I., Ruysschaert, J. M. and O'Shea, P. (1999) J.    Biol. Chem. 274, 29951–29959    the teaching of all of which is incorporated herein by reference.    The technique has been demonstrated to possess the additional virtue    that it may be applied to living cells as well as to model membrane    systems.

EXAMPLE 1

Interaction of Designer Peptides with Human Cells Possessing MembraneReceptor

Human umbilical endothelial cells were labelled with FPE and challengedwith the indicated amounts of BVS1 peptides. Similar data were obtainedwith Molt4 T lymphocytes at 10⁵ ml⁻¹ suspended in 280 mM sucrose, 10 mMhepes at pH7.4. Fluorescence was recorded at 518 nm after excitation at490 nm.

Two separate experiments are shown and offset in the time dimension forclarity. Peptides were added as indicated by the arrows. The BVS1peptide was added in both experiments. In the second trace, however,addition of BVS1 was preceded by the addition of 300 nM IL-8.

Example 1 illustrates that the BVS1 peptide clearly binds to a receptorwith the same characteristics located in a number of cell typesincluding human umbilical vein endothelial cells (HUVECS) and culturedhuman white blood cells. Also illustrated is the fact that this receptorappears to bind the molecule known as interleukin-8 (IL-8) and, onceoccupied with this molecule, the receptor is unable to bind the BVS1.This may indicate that the receptor targeted by BVS1 is very close tothe IL-8 receptor or is in very close proximity to it. The IL-8 receptoris known to exert its action via g-protein coupled signalling processes(Biochem J (1992) 282, 429–34, “G-protein activation by interleukin-8and related cytokines in human neutrophil plasma membranes”, Kupper R W,Dewald B, Jakobs K H, Baggiolini M, Gierschik P) and these are locatedwithin the membrane microdomain structures referred to as PMLMs.

EXAMPLE 2

Interaction of Designer Peptides with Purified Isolated Receptor

FIG. 2 illustrates the interactions of BVS1 with a receptor isolatedfrom the HUVECS plasma membrane and reconstituted into artificialmembranes labelled with FPE. At the arrows, 5 μM BVS1 was added. Twoseparate experiments are shown and offset in the time dimension forclarity. In the second trace, however, addition of BVS1 was preceded bythe addition of 5 μM of human serum albumin.

Example 2 illustrates that the BVS1 peptide clearly binds to a receptorisolated from human umbilical vein endothelial cells (HUVECS). Thereconstitution of the purified receptor was achieved essentially by amodification of the protocol outlined in Methods Enzymol (1986), 126,78–87, “Functional reconstitution of proton-pumping cytochrome-c oxidasein phospholipid vesicles”, Muller M, Thelen M, O'Shea P, Azzi A. Thefluorescent increase is indicative of interactions between the designerpeptide BVS1 and a reconstituted receptor protein molecule normallylocalised in the PMLMs, in this case a protein involved in the uptake ofalbumin. Also illustrated is the fact that this receptor appears to bindhuman serum albumin and, once occupied with this molecule, the receptoris unable to bind the BVS1.

EXAMPLE 3

Interaction of Peptido-Mimetic with Purified Isolated Receptor

FIG. 3 illustrates the interaction of polysulfonylnaphthylurea (PSNU)with a receptor isolated from the plasma membrane of HUVEC,reconstituted into artificial membranes and labelled with FPE At thearrow, 2 μM PSNU was added.

These data clearly demonstrate that interactions take place between theadded polysulphonyl napthylureas with an albumin receptor proteinmolecule reconstituted into model membranes and labelled with FPE in asimilar manner to Example 2. These receptors are normally localised inthe PMLMs and may be involved in transcellular transport ofmacromolecules. Similar results can be obtained with peptidesBVS17–BVS20 or similar mimetics. Reduced/no effects are obtained usingmodel membranes in the absence of any PMLM intrinsic components or withpeptides/mimetics that possess no binding properties to PMLM receptors.

EXAMPLE 4

Transcellular Transport of Indicator Macromolecules is Augmented byReagents Listed in Examples 1–3

Transport of horse radish peroxidase (HRP) across a cellular membranewas measured using the apparatus shown schematically in FIG. 4, with theaddition of biovectors (BVS1 etc). The apparatus comprises upper andlower compartments (1, 2 respectively) separated by a filter 3 on whichthe cells 4 are grown. HRP and biovectors are added to the medium in theupper compartment 1, and HRP is assayed in the lower compartment 2.

The results are shown in Table 1 and show that enhanced transport ofmacromolecules such as HRP by as much as 20 fold increment (depending onthe chemical nature of the biovector) over control studies involvingaddition of HRP can be achieved using biovectors (eg designer peptidessuch BVS1). This increase is larger than the effect of the polysulphonylnapthylureas studied.

TABLE 1 Specific resistance Across Baseline Facilitated monolayertransport transport System Ohms/cm⁻² nM/min⁻¹ X Fold +5 μMBVS1 >3500.003 15  +5 μM PSNU >350 0.003 3 +5 μM PSNU >350 0.003 3 then +5 μMBVS10.003 3/0 +5 μMBVS5 >350 0.003 22  +5 μMBVS9 >350 0.003 7 +5μMBVS16 >350 0.003 9

EXAMPLE 5

Transcellular Transport Across Monolavers of Caco2 Cells Augmented byBVS1 Peptide

Caco2 cells were cultured (passage 17–30) with standard methods and usedat ca 21 days. Cells were plated at a density of ca. 50×10⁻³ cm⁻² onTranswell® filters (2–4 μm, mean pore size).

The viability of cells for transport was assessed on the basis of thetranscellular electrical resistance and values >350 Ohms cm⁻² wereassessed as viable.

Transcellular transport was determined as above for the HUVECs studies.In the absence of BVS1 transcellular transport was determined to be ca0.001 nM min⁻¹. Following treatment with 5 μM BVS1, transport wasobserved to be elevated to 0.02 nM min⁻¹.

Example 5 is consistent with Example 4 in that an increase in thetransport of HRP was observed following treatment of a cell monolayerwith BVS1. In this case the Caco2 cell line was utilised as it hasbecome an accepted standard model system for gastrointestinal systems.

1. A method of modifying transport processes across an endothelial,epithelial or mesothelial membrane which method comprises administeringto the membrane an effective amount of a peptide compound consisting ofthe amino acid sequence: AVGIGALFLGFLGAAG (SEQ ID NO:1).
 2. A method asclaimed in claim 1, wherein the compound enhances transport across themembrane.
 3. A conjugate comprising a therapeutically active agentconjugated to a peptide compound consisting of the amino acid sequence:AVGIGALFLGFLGAAG (SEQ ID NO:1).